Liquid fluid bed chromatography using conglomerates of controlled density

ABSTRACT

A fluidized bed chromatographic process for the purification and binding of molecules in a liquid to an active substance covalently bound to chromatographic adsorbent particles. The adsorbent particles are formed of a porous composite material having pores allowing access to the interior thereof and consisting of at least two density controlling particles of low or high density, or both, and a matrix formed by consolidating at least one conglomerating agent. The density controlling particles are dispersed in the matrix. The adsorbent particles have a relative density with respect to the liquid which is less than 0.95 and greater than 1.1, and they have a particle size within the range of 50-750 μm. The relative density and particle size range of the adsorbent particles are selected to provide desired floatation/sedimentation properties of the adsorbent particles in the liquid in the fluidized bed process.

1. BACKGROUND OF THE INVENTION

1. The Technical Field

The invention relates to a conglomerate of controlled relative densityfor containing or carrying at least one active substance to be used in afluid; methods of preparing such a conglomerate; and the use of such aconglomerate as a solid phase matrix, carrier, or substrate material ina fluid bed rector, or in a batch reactor; as a carrier of substancesfor sustained release; as a food material, medical, and vaccine forfish, or other animals living in water; as a material for treating wastewater and polluted waters; and as a material for treating polluted watersuch as oil poluted sea water.

Further, the invention relates to a method of distributing a fluid inthe fluid bed of a fluid bed reactor; and a fluid bed reactor using sucha method.

In the present context the expression "conglomerate" is intended todesignate a composite of basic particles, which may comprise particlesof different types and sizes, held together by conglomerating agents.Conglomerates may be of various sizes, and shapes and should preferablyexhibit various degrees of mechanical rigidity depending on theapplication. Further, conglomerates may be chemically active or may bechemically inactive under the conditions applied.

The expression "conglomerate of controlled relative density" is intendedto designate a conglomerate or a conglomerate particle for which inparticular the basic particles are chosen in predetermined amounts toprovide a certain relative density of the conglomerate with respect tothe fluid in which an active substance or another constituent of theconglomerate is to be used so that the floatability or sedimentation,respectively, is controlled. Thus, conglomerates according to theinvention are intentionally designed with respect to the density of themedium for their particular purpose of application, including properconsideration of the influence of their sizes on their floating orsedimentation properties. In other media, e.g. during preparation orduring storage under e.g. dry conditions, the conglomerate may have adensity different from than that in the fluid medium of use, such fluidsmay be liquids or gases.

In the present context the expression "active substance" should be takenin a very broad sense comprising agents having desired properties fortheir particular purpose of application, e.g. adsorbents, ligands,reagents, enzymes, catalysts; natural substances and substrates, cellaggregates; or nutritional matter for animals living in water; entrappedin or chemically, e.g. covalently, ionically, photochemically, etc.bound to the conglomerate of controlled density.

Carrier materials for carrying at least one active substance are used ina wide variety of applications in chemical and biological processes,such as production and manufacturing of chemical or pharmaceuticalproducts, e.g. for carrying catalysts in liquid phase oil conversiontechnology, for carrying enzymes for modifying synthetical products,e.g. enzymes such as proteases, invertases, amidases and ring formingenzymes for synthesis of lactones, and carboxypeptidase for synthesis ofpeptides using solid-phase techniques; fermentation and cell growth,e.g. for carrying cells or substrate; waste water purification, e.g. forcarrying enzymes and/or microorganisms, catalysts or adsorbents;chromatographic processes, e.g. high performance liquid chromatography,gel filtration, ion exchange and affinity chromatography, e.g. forcarrying adsorbents; diagnostic processes, e.g. for carrying adsorbentsfor blood purification, dye chromatographic processes for albuminepurification; and prophylactic processes, e.g. for carrying immobilizedantibodies or antigens in extracorporal circulations for removal ofantigens or antibodies, bacterial toxins or other toxins, and autoimmunediseases.

2. Prior Art Disclosure

There are numerous disclosures in the prior art concerning particlesprepared from organic and inorganic materials. However, carrierscomprising conglomerates of controlled relative density with respect tothe fluid of use carrying or for carrying at least one active substancehave apparently never been disclosed.

Kuraray Co., Ltd., C.A. 98:157436t discloses beads, particles, fibres,sheets, and tubes of glass, activated carbon, silica, alumina or highmolecular weight substances coated with copolymers of acrylates andcarboxylic acids or amines to form selective adsorbent carriers orsupports for use in selective electrodes or in column chromatography.

Sakuma et al., C.A. 111:74363c, disclose glass or polymer spheres coatedwith hydroxyapatite for use as a stationary phase for columnchromatography.

EP-A-0266580 discloses a method for coating solid particles with ahydrophilic gel preferably agarose for various separating processes inpacked columns based on adsorbent groups, e.g. ion exchanging groups,hydrophobic groups, or groups with biospecificity chemically bound to agel. Such coating may be provided by mixing hydrophilic solid particleswith a gel-forming substance above the gelling temperature in which eachindividual particle is coated, separated from each other, and cooledbelow the gelling temperature, essentially to stabilize the particlesagainst the high pressure in e.g. HPLC applications

Generally, all of the above mentioned coated particles are provided bycoating individual particles made of the same material and having thesame density.

U.S. Pat. No. 4,698,317 discloses hollow microspherical glass particleshaving open pores, and being prepared by spray thermal decomposition ofa solution, in an aqueous organic solvent, wherein the water contentpromotes open pore formation.

U.S. Pat. No. 2,797,201 discloses substantially spherical, hollowparticles having a "thin, strong skin" being prepared by thermaltreatment of droplets of a solution of a film forming material, e.g. anorganic polymer such as a phenolformaldehyde resin, and optionallyfurther containing a "blowing agent", i.e an agent generating gas at theelevated temperature of the thermal treatment.

GB 2151601B discloses porous hollow particles of an inorganic materialand a composite material comprising such particles supporting a selectedsubstance such as a chromatographic organic gel. The porous hollowparticles may be formed by coating a fugitive core material, e.g.organic resin beads or alginate spheres, with inorganic material, andthen heating to remove the fugitive core material. Further, GB 2151602Bdiscloses closely similar particles wherein a magnetic material, such asferric oxide, nickel oxid or cobalt oxide, is incorporated in theinorganic shell of the particle.

The 3M Corporation (USA) markets a number of types of substantiallyimpermeable, hollow micro-spheres of silicious material. For examplesynthetically manufactured soda-lime borosilicate glass micro-spheresmarketed by 3M in a variety of size fractions. Also, permeable hollowspheres of siliceous material derived from fly-ash are provided byFillite Ltd., Runcorn, England. However, none of the commerciallyavailable micro-spheres are conglomerates of controlled relative densityaccording to the invention.

EP-A-0021563 discloses a material suitable for thermosetting whichincludes a collection of hollow particles adhesively mixed with athermosetting resin and which material may be converted by thermosettinginto a fused solid mass having a density not greater than 0.5 g/cm³.

GB-A-2196252 discloses an oral, solid, pharmaceutical dosage formcomprising conventional matrix binders including starch and cellulose,or their derivatives, and a pharmaceutically acceptable weighting agent,including inorganic compounds such as salts, oxides, or hydroxides of ametal, e.g. barium sulphate or ferrous oxide, suitable for oraladministration to humans and for controlled release of apharmaceutically active ingredient into the stomach. The controlledrelease unit may have any chosen density from about 2 g/ml to about 6g/ml and may in case of a conventional pellet have a size from about 1to about 1.4 mm, and in case of a tablet a size above 10 mm. Nothing isdisclosed nor suggested about non-solid i.e. permeable or porousconglomerates of controlled relative density according to the invention.Furtermore, the described pharmaceutical dosage form consists of solidparticles comprising a binder and a weighting agent soluble in gasticfluid which makes the pellet or tablet disintegrate shortly afteringestion.

Generally, for a large number of applications, the active substance tobe used in a fluid may only temporarily be available or accessible atthe right places in the fluid. Thus, for inert carrier particlescarrying active substances and often showing large variations indispersion properties, e.g. sedimention or floatation, the activesubstances may be carried in an uncontrolled manner e.g. down- orupwards in relation to the fluid depending on the relative density ofthe carrier.

In fluid bed reactors partially solving the problems of packed bedcolumns, i.e. the problems of suspended matter clogging up thesolid-phase bed which increases the back pressures and compresses thebed disturbing the flow through the bed, the carrier particles arecarrying the active substance in a free, fluid phase by applying a flowhaving an opposite direction to the direction of the relative movementof the carrier. Thus, carrier particles having a density larger than thefluid and moving downwards due to gravity may be kept in a free, fluidphase by an upwards flow of fluid. Also, carrier particles having adensity less than the fluid and thus moving upwards may due to buoyancybe kept free, fluid phase by a downwards flow of fluid.

For fluid bed solid-phase chemical processes, the density of thesolid-phase carrier particle is very important in controlling bedproperties. However, up to now, the design of solid-phase carrierparticles has been limited by the available material.

Generally, particles may either be designed to be impermeable to thefluid, in which case the available surface area per unit volume issmall; or particles may be designed to be permeable to the fluid, inwhich case the material chosen has to have the correct density per se.Unfortunately, the most interesting materials for many applications,e.g. materials such as natural and synthetic polysaccharides like agar,alginates, carrageenans, agarose, dextran, modified starches, andcelluloses; synthetic organic polymers and copolymers typically based onacrylic monomers used for chromatographic purification of proteins inpacked bed columns are not of suitable density per se. Therefore, thesematerials are difficult to apply in fluid bed reactors.

However, certain types of organic polymers and certain types of silicabased materials may be produced to provide carrier particles of suitabledensity, but such carriers may not at the same time be suitable activesubstances, e.g. for protein purification procedures, where suchmaterials may provide low permeability, non-specific interactions anddenature bound proteins. Further, for such polymers, it may be difficultand expensive to design derivatisation schemes for affinitychromatography media. Also, certain types of permeable silica particleshave been used for fluid bed applications. However, the properties ofthese materials are far from optimal. Thus, the materials are instableat pH above 7, fragile to shear forces, and provide non-specificinteractions.

U.S. Pat. No. 4,032,407 discloses a tapered bed bioreactor applyingimmobilized biological catalysts or enzymatic systems on fluidizableparticulate support materials consisting of coal, alumina, sand, andglass, i.e. materials heavier than the fluid.

EP-A-0175568 discloses a three phase fluidized bioreactor processcomprising purifying effluents in a three phase fluidized bed comprisingsolid particles being made by mixing a binder with an inorganic materialbesed on aluminum silicate, granulating the resulting mixture, andfiring the granules to sinter them. The specific gravity of the sinteredgranules is adjusted to fall into a specific range from 1.2 to 2.0 byvarying the mixing ratio of inorganic powdery materials based onaluminum and binders, said sintered granules having a diameter from 0.1to 5 mm.

EP-A-0025309 discloses a downflow fluid bed bioreactor applying biotaattached to carrier particles consisting of cork, wood, plasticparticles, hollow glass beads or other light weight material and havinga specific gravity which is less than that of a liquid sprayed onto theupper part of a fluid bed of suspended carrier particles and conducteddownward through the bed.

These three disclosures describe particulate support materials to whichthe attachment of the active substance is restricted to the surface ofthe particles limiting the amount of active substance to be obtained perunit volume compared to particles allowing the active substance to beattached within the particle. Thus, in many applications, it isimportant to have specifically designed particles able to carry as largean amount of active substance per unit volume as possible whichparticles are not available in the prior art.

Thus, in great many applications of active substances in fluids, thereis a need for materials of controlled relative density carrying or forcarrying active substances in the fluids.

Further, a disadvantage is that the fluid is distributed in the fluidbed of a fluid bed reactor by spraying whereby channels are formed inthe bed by the impinging fluid rays. International ApplicationPublication No. WO81/02844 discloses a multi-lagered filter mediumcomprising particles formed of hollow silica beads distributed in amatrix of cured cement having a uniform specific gravity in the rangefrom 1.02 to about 1.5; said particles being adapted for use assucessive layers in a deep bed filter to promote agitation and scrubbingof the particles and to separate the particles more efficiently duringbackwash, i.e. for a use which does not involve an active substance. Theparticles are prepared by casting a slurry of hollow silica beadsdispersed in a binder material such as cement; curing the casted slurryto a self-sustaining state; and cutting the cured casted slurry intopolygonal granules; said granules then being completely cured. Nothingis indicated or suggested about using an organic binder material, andproviding the granules with an active substance.

EP-A-0005650 discloses an up-flow fluid bed reactor having fluidizingfluid flow distributors at the bottom thereof providing flow paths toavoid turbulent effects. Besides requiring complicated flow paths, adisadvantage of such a distributor is that it may be clogged byparticulate matter.

U.S. Pat. No. 4,142,969 discloses an oleospecific hydrophobiccomposition comprising an intimate mixture of expanded volcanic glassconsisting of perlite, a cellulose fiber, and a water repellent sizingconsisting of asphalt; and a method of sorbing oleaginous compounds e.g.in selectively removing oil from the surface of water. The constituentsare incorporated into a homogeneous product by a wet process, dried inan oven until mixture essentially all moisture has been removed, andthen ground up into a fluffy low density material. Nothing is disclosednor suggested about controlling the density of the composition byincorporation of high or low density particles.

2. DISCLOSURE OF THE INVENTION

(a) Conglomerates

It is the object of the present invention to provide a conglomerate forcarrying at least one active substance to be used in a fluid and havinga controlled relative density with respect to the fluid.

Particularly, it is the object of the present invention to provide sucha conglomerate which avoids the disadvantages of known carriermaterials, e.g. the problems of uncontrolled sedimentation or floatationof active substance and/or its carrier, the poor selectivity andcapacity of carriers having immobilized active substances, and themissing possibilities of simultaneously designing and controlling theproperties of the active substance and carrier

According to the invention this is fulfilled by providing a conglomeratehaving controlled relative density for containing or carrying at leastone active substance to be used in a fluid, characterized in that itcomprises predetermined amounts of:

a) basic particles selected from the group consisting of low densityparticles having a density providing floatation and high densityparticles having a density providing sedimentation of the conglomeratein said fluid;

said basic particles being dispersed in

b) a matrix formed by consolidating at least one conglomerating agentselected from the group consisting of natural and synthetic organicmonomers and polymers; and

c) optionally at least one active substance bound to, entrapped in,carried or constituted by said matrix.

It surprisingly turns out by providing a conglomerate comprising

a) basic particles selected from the group consisting of low densityparticles having a density providing floatation and high densityparticles having a density providing sedimentation of the conglomeratein said fluid;

said basic particles being dispersed in

b) a matrix formed by consolidating at least one conglomerating agentselected from the group consisting of natural and synthetic organicmonomers and polymers;

and all constituents in suitable predetermined amounts, that it isensured that the relative density of the conglomerate can be controlledwithin suitable limits for the application In the fluid where it is tobe used.

Further, providing the conglomerate with optionally at least one activesubstance bound to, entrapped in, carried or constituted by said matrix,it is ensured that the conglomerate is or it can be provided with adesired properties such as chemical or biological selectivity orcapacity. Also, it is ensured that both the properties of the activesubstance and the conglomerate can be controlled simultaneously.

Particularly, a conglomerate not comprising the active substance as suchmay be prepared separately in order to provide a material of controlledrelative density which can be treated to comprise the active substance.

Conglomerates may further comprise other substances such as additives,fillings, softeners, etc., and may be designed to e.g. controlledrelease (also known as sustained release, slow release or "retard"release) of a desired substance from a conglomerate depending on thechoice of conglomerating agent, and possibly comprising a suitablesurface coating, e.g. of a material such as the ones mentioned for theconglomerating agent, having diffusion or permeability characteristicsappropriate to the gradual release of the substance in question from theconglomerate.

In its broadest aspect the density may be controlled by selecting basicparticles from a group of particles consisting of particles of very lowdensity providing floatation of the conglomerate, particularly hollowand impermeable particles having shells of suitable material andproperties, however, non-hollow particles may be chosen whenappropriate; and particles of very high density, e.g. particles based onsuitable heavy elements or compounds providing sedimentation of theconglomerate.

Generally, the invention provides a new type of carrier particlescomprising conglomerates of controlled relative density, selectivity,and capacity in terms of controllable interior surface areas andmaterials e.g. materials having specific chemical and/or mechanicalproperties. Thus, compared with known carrier particles for fluid andpacked bed reactors, conglomerates according to the invention cansurprisingly be designed to have a number of advantages not previouslyobtained.

Conglomerates according to the invention e.g. in form of carrierparticles can be designed to have a controlled relative densityindependent of the active substances and the conglomerating agents;heavy particles can be made light, and vice versa, within a wide rangeof particle sizes; the density can be controlled within very broadlimits, e.g. the density of a known material can be controlled for aspecific application; the volume percentage of the conglomerating agentcan be controlled according to the application; the total size of thefinal carrier particle can be controlled contrary to known particleshaving uncontrollable sizes for specific densities suitable forparticular rising and falling velocities; further, conglomeratesaccording to the invention have a relative larger capacity, i.e. alarger accessible volume, compared to e.g. known impermeable carrierparticles; also, in preparing such known impermeable carrier particles,the active substances to be applied are limited, e.g. limited tosubstances that can be attached to the particle surface. However, forconglomerates, both the impermeable carrier particles and the activesubstances may be included, e.g. in form of entrapped particles orsubstances.

Also, contrary to known particles having a given mechanical strength anddensity, the elasticity and the mechanical strength of a conglomeratecan be controlled independent of the density. Further, pore sizes ande.g. biocompatibility can be controlled independently of the density inorder to allow access to the interior of the conglomerate and to avoiddenaturation e.g. of proteins.

(b) Methods of Preparation

Preparation of conglomerates according to the invention may be obtainedby various methods known per se, e.g. block polymerisation of monomers;suspension polymerisation of Monomers; block or suspension gelation ofgelforming materials, e.g. by heating and cooling (e.g. of agarose) orby addition of gelation "catalysts" (e.g. adding a suitable metalion toalginates or carrageenans); block or suspension cross-linking ofsuitable soluble materials (e.g. cross-linking of dextrans, celluloses,starches or gelatines, or other organic polymers with e.g.epichlorohydrine or divinyl sulfon); formation of silica polymers byacidification of silica solutions (e.g. block or suspension solutions);mixed procedures e.g. polymerisation and gelation; spraying procedures;and fluid bed coating of basic; particles.

Thus, for particularly preferred embodiments according to the invention,conglomerates may be obtained by cooling emulsions of basic particlessuspended in conglomerating agents in heated oil solvents; or bysuspending basic particles and active substance in a suitable momoner orcopolymer solution followed by polymerisation.

"Preparation by Gelation/Polymerisation in the Emulsified State"

In another aspect the invention provides a method of preparing aconglomerate according to the invention, comprising

a) mixing basic particles selected from the group consisting of lowdensity particles having a density providing floatation, and highdensity particles having a density providing sedimentation of theconglomerate in the fluid, said particles preferably impermeable to thefluid,

at least one conglomerating agent made of a material consisting ofnatural and synthetic organic monomers and/or polymers selected from thegroup consisting of:

i) natural and synthetic polysaccharides and other carbohydrate basedpolymers, including agar, alginate, carrageenan, guar gum, gum arabic,gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum,agaroses, celluloses, pectins, mucins, dextrans, starches, heparins,gelatins, chitosans, hydroxy starches, hydroxypropyl starches,carboxymethyl starches, hydroxyethyl celluloses, hydroxypropylcelluloses, and carboxymethyl celluloses;

ii) synthetic organic polymers and monomers resulting in polymers,including acrylic polymers, polyamides, polyimides, polyesters,polyethers, polymeric vinyl compounds, polyalkenes, and substitutedderivatives thereof, as well as copolymers comprising more than one suchorganic polymer functionality, and substituted derivatives thereof; and

iii) mixtures of these;

said active substance, if present, in predetermined amounts optionallyheated;

b) emulsifying said mixture in a suitable solvent;

c) consolidating said conglomerating agent by a suitable means such asgelation by heating/cooling, polymerisation of monomer or monomermixtures, non-covalent or covalent cross-bonding; and

d) isolating and washing of the conglomerate.

"Preparation by Gelation/Polymerisation in the Block State"

In still another aspect, the invention provides a method of preparing aconglomerate according to the invention, comprising

a) mixing basic particles selected from the group consisting of lowdensity particles having a density providing floatation, and highdensity particles having a density providing sedimentation of theconglomerate in the fluid, said particles preferably impermeable to thefluid,

at least one conglomerating agent made of a material consisting ofnatural and synthetic organic monomers and/or polymers selected from thegroup consisting of:

i) natural and synthetic polysaccharides and other carbohydrate basedpolymers, including agar, aliginate, carrageenan, guar gum, gum arabic,gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum,agaroses, celluloses, pectins, mucins dextrans, starches, heparins,gelatins, chitosans, hydroxy starches, hydroxypropyl starches,carboxymethyl starches, hydroxyethyl celluloses, hydroxypropylcelluloses, and carboxymethyl celluloses;

ii) synthetic organic polymers and monomers resulting in polymers,including acrylic polymers, polyamides, polyimides, polyesters,polyethers, polymeric vinyl compounds, polyalkenes, and substitutedderivatives thereof, as well as copolymers comprising more than one suchorganic polymer functionality, and substituted derivatives thereof; and

iii) mixtures of these;

said active substance, if present, in predetermined amounts in asolvent; and

b) consolidating said conglomerating agent by a suitable means such asgelation by heating/cooling, polymerisation of monomer or monomermixtures, non-covalent or covalent cross-bonding; and

c) disintegrating the block of conglomerate; and

d) segregating the particles, and washing the segregated conglomerate.

Thus, e.g. for polysaccharides such as agarose and agar, i.e. materialsmelting at high temperatures and solidifying at low temperatures, theconglomerating means is by heating/cooling. Further, for acrylderivatives and other monomers or mixtures of these, the conglomeratingmeans can be selected from a group consisting of:

a) addition of polymerisation catalyst;

b) heating;

c) illumination with light; and

d) irradiation with ionizing radiation.

Particularly for heavily charged polysaccharides and polymers such asalginates and carrageenans, the conglomerating means is non-covalentcross-bonding by addition of a suitable metal ion However, forpolysaccharides in general, e.g. cellulose and its derivatives, andpolymer containing e.g. amino, hydroxyl, thiol, and carboxy groups, theconglomerating means is covalent cross-bonding by addition of a suitablecross-bonding agent, e.g. epichlorohydrine, divinyl sulfon,bisepoxyranes, dibromopropanol, glutaric dialdehyde, diamines, and otherbifunctional agents.

Also, the above mentioned conglomerating means may be combined inspecific cases such as the preparation of conglomerates ofagarose-acryl-derivatives and cross-bonded mixtures of agarose anddextran.

Further, in the above mentioned block polymerisation, the segregationstep of the polymer block may be obtained by methods known per se, e.g.by granulation and sieving.

(c) The Use of Conglomerates

"Solid Phase Matrix, Carriers, or Substrate Materials"

The invention also relates to the use of conglomerates according to theinvention as a solid phase matrix, carrier, or substrate material in aprocedure selected from the group consisting of:

chromatographic procedures applying non-packed columns including liquidchromatography, ion-exchange chromatography, and biospecific affinitychromatography such as immunosorption and protein A chromatography, andgroup specific affinity chromatography such as hydrophobic, thiophilic,dye, lectin, and metal chelate chromatography;

filtration of a fluid medium;

absorption of at least one selected substance present in a fluid medium;

heterogeneous catalysis of a reaction taking place in a fluid medium;

immunochemical procedures, including immunosorbtion;

solid-phase synthesis, including solid-phase peptide and proteinsynthesis, and solid-phase oligonucleotide synthesis;

microbiological procedures;

enzyme reactor procedures;

carriage, on the outer or the interior surface of the particles,optionally after a suitable surface treatment of live cells selectedfrom cells of human, animal, plant, fungal and microorganism origin.

Examples of enzyme reactor procedures are:

(i) "confinement immobilization" procedures making use of an enzyme(e.g. in the form of an enzyme solution) which is contained within thethrough-going pores and/or internal cavities of a permeableconglomerate, and which is prevented, as described earlier, above, fromescaping from the conglomerate by the presence of a suitable surfacecoating having diffusion or permeability characteristics such that thedesired enzyme substrate(s) and resulting reaction product(s) maymigrate through the coating;

(ii) "solid-phase covalent immobilization" procedures making use of anenzyme which is covalently bound, via appropriate functionalities,within the conglomerate, the resulting conglomerate optionally beingsubjected to a surface treatment to provided a coating of the typementioned in (i) above.

Such procedures might be employed, for example, in the production ofhigh-fructose syrups from sucrose molasses, using a permeableconglomerate containing a suitable "confinement immobilized" or"solid-phase covalently immobilized" sucrase.

"Fluid Bed Reactors"

Generally, a fluid bed reactor may comprise a vertical reactor with aninlet, an outlet, a fluid bed of particles, and a fluid. The fluid isintroduced at the inlet and dispersed, optionally through a gas head incase of down-flow reactors, on the bed of particles which are suspendedand fluidized by the fluid. The fluid it conducted through the bed and apool of reacted and/or unreacted fluid is let out at the outlet.

Down-flow fluid bed reactors have fluid inlet at the top of the reactorand fluid bed particles of specific gravity less than that of the fluid.

Up-flow fluid reactors have fluid inlet at the bottom of the reactor andfluid bed particles of specific gravity larger than that of the fluid.

The suspended particles may be reactive or may carry immobilizedreactive components selected for solid phase chemical or physicalprocesses with one or more components of the fluid in procedures such asenzymatic reactions; fermentation; ion-exchange and affinitychromatography; filtration; adsorption; catalysis; immunosorption;solid-phase peptide and protein synthesis; and microbiological growth ofmicroorganisms.

It is an object of the invention to provide the use of conglomeratesaccording to the invention in solid-phase chemical processes such asheterogeneous chemical reactions in continuous fluid bed reactorsparticularly for separation of proteins.

This is fulfilled by providing the use of a conglomerate or particles ofa conglomerate according to the invention, or a conglomerate prepared bya method of preparing a conglomerate according to the invention, as asolid phase matrix, carrier, or substrate material in a fluid bedreactor.

"Distribution of Fluid in the Fluid Bed of a Fluid Bed Reactor"

Generally, in carrying out solid phase chemical or physical processes ina fluid bed reactors it is desired to have an even and smoothdistribution of fluid in the fluid bed.

To provide this, it is known to use a fluid flow distribution by meansof a distribution plate which, however, does not avoid the formation ofchannels in the fluid bed, jsut as unwanted turbulence occurs.

According to the invention, it has surprisingly turned out thatagitation of the part of the fluid bed proximal to the inlet of thefluid divides the fluid bed into

i) an agitated zone having vigorously moving particles; and

ii) a non-agitated zone;

said non-agitated zone adjoining said agitated zone in a sharp interfaceacross which there is an even distribution of fluid flowing into thenon-agitated zone with minimal or no turbulence.

Further, it is obtained that unwanted mixing of products and reactants,and unwanted wear of the bed particles are reduced.

Therefore, in another aspect, it is the object of the present inventionto provide a method of distributing a fluid in the fluid bed of a fluidbed reactor such that the fluid is distributed evenly with minimal or noturbulence in the fluid bed.

According to the invention, there is provided a method of distributing afluid in the fluid bed of a down-flow fluid bed reactor comprising avertical reactor with an inlet, an outlet, and a fluid bed of particles,wherein

a) the particles and fluid proximal to the fluid inlet are agitated todivide the fluid bed into

i) an agitated zone having vigorously moving particles, and

ii) a non-agitated zone;

said non-agitated zone adjoining said agitated zone; and

b) the extent of said agitated zone is determined by a degree ofagitation selected within a range from

i) a degree of agitation providing agitation only in the uppermost partof the fluid bed,

ii) to a degree of agitation providing agitation of the particlesthroughout the fluid bed.

Also, for chromatographic applications, the dispersion of eluant isreduced, i.e. the width of the eluation band is reduced. Also, forenzymatic and fermentation type reactions, enzyme inhibition is reducedas the intermixing of products and substrates is reduced. Further,formation of channels in the fluid bed is minimized.

The invention provides a similar method for an up-flow fluid bed reactorin which the extent of the agitated zone is determined by thesedimentation of the fluid bed particles instead of the buoyancy of theparticles as in case of the down-flow fluid bed reactor.

In a particularly preferred embodiment, the invention provides suchmethod of distribution a fluid in the fluid bed of a fluid bed reactorwherein the particles consists of a conglomerate according to theinvention.

The position of the sharp interface is controlled by the degree ofagitation which is selected for a given flow of fluid, viscosity, andbuoyance/sedimentation of the particles.

Agitation can be effected by any agitation means, including mechanicalagitation mean or gas injection mean. In case of mechanical agitationmeans, it is preferred that the agitation is provided by stirring with amechanical stirrer which does not form a vortex in the fluid.

Fluid bed particles can be different or of the same type for both theagitated zone and the non-agitated zone.

In a preferred embodiment, the agitated zone may comprise inertparticles of slightly different specific gravity than the particles ofthe non-agitated zone. In this case, the inert particles positioned inthe agitation zone solely participate in the distribution of fluid inthe fluid bed, and not in the solid phase processes. These processestake place in the non-agitated zone by specifically designed particleshaving both controlled density and controlled chemical reactivity,capacity, etc.

It is further the object of the present invention to provide a fluid bedreactor using such a method of distributing the fluid in the fluid bed.

According to the invention, there is provided a down-flow fluid bedreactor comprising a vertical reactor vessel with an inlet, an outlet, afluid bed of particles, and agitation means, characterized in that theagitation means is located near or in the fluid bed proximal to thefluid inlet.

Also, according to the invention, there is provided an upflow fluid bedreactor comprising a vertical reactor vessel with an inlet, an outlet, afluid bed of particles, and agitation means, characterized in that theagitation means is located near or in the fluid bed proximal to thefluid inlet.

In a preferred embodiment, both down-flow and up-flow fluid bed reactorscomprise a fluid bed the particles of which consist of a conglomerateaccording to the invention.

Generally, compared to packed bed techniques, fluid bed techniques, e.g.to be used in fluid bed chromatography, are better suitable to largescale primary purification of proteins as the steps of centrifugationand filtration can be avoided. Thus, the fluid bed techniques can beused immediately following the production of the protein, e.g. directlyapplying the produced extract or fermentation fluid to fluid bedpurification and conversion. Accordingly, using conglomerates accordingto the invention in fluid bed techniques, several advantages such as thecontrol of the density, and the choice of materials to design thechemical and/or mechanical properties of the carrier particles, e.g.including cheaper basic materials, are obtained.

"Batch Reactors"

Another common way of performing solid phase reactions e.g. adsorptionof at least one selected substance from a fluid medium in non-packedcolumns is by the way of adsorption in a batch reactor or batch columne.g. a simple vessel wherein the conglomerate particles are mixed withthe liquid in a one-step procedure.

In another aspect, the invention provides the use of a conglomerate orparticles of a conglomerate according to the invention, or aconglomerate prepared by a method of preparing a conglomerate accordingto the invention, as a solid phase matrix, carrier, or substratematerial in a batch reactor.

Thus, an enhanced speed of separation of conglomerate particles in abatch reactor process can be obtained by choosing an optimallycontrolled relative density of the particles.

A particularly preferred way of separating the conglomerate particles isto pump the liquid containing the particles through a particlecollection vessel which traps the particles e.g. by letting theparticles with relative low density float up-wards in the vessel andletting the liquid flow through.

"Carriers of Substances for Sustained Release"

In another aspect the invention provides conglomerates of controlledrelative density to be used for sustained release of a desired substancefrom a conglomerate depending on the choice of conglomerating agent andspecific application. Thus such conglomerates may comprise a suitablesurface coating, e.g. of a material such as the ones mentioned for theconglomerating agent. Further, the conglomerates have diffusion orpermeability characteristics appropriate to the gradual release of thesubstance in question.

"Carriers of Substances for Animals Living in Water"

In still other aspects the invention provides conglomerates ofcontrolled relative density to-be used-in foods, medicals, and vaccinesfor fish, or other animals living in water, which substances may beadministered most efficiently if they do not sediment on the bottom,where they may be lost or eaten by other animals.

"Carriers of Substances for Waste Water Treatment"

In still another aspect the invention provides conglomerates ofcontrolled relative density for waste water treatment materials, whereinthe active substance is a microbial cell, an enzyme, a catalyst oranother treatment material purifying or at least partly purifying thewater. Floating particles comprising waste water treatment material mayhave the advantage of oxygen being available for e.g. microorganismsgrowing in the interior of the conglomerate.

"Carriers of Substances for Treating Oil Polluted Waters"

In still another aspect the invention provides conglomerates ofcontrolled relative density for treating oil polluted waters, whereinthe active substance is a microbial cell capable of breaking down andfeed on oil, or is a catalyst for breaking down oil and oil emulsions.The active substance may also comprise materials suitable for selectivehydrofobic adsorption of oil, such as certain type of surface treatedperlite. Thus, conglomerates for treating oil pollution may have theadvantage of being designed to be in close contact with the oil or theoil emulsions at the water surface, e.g. confined within a certain areaof the surface by means of pontoons.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by reference to theexamples given below and to FIG. 1-7, wherein

FIG. 1A shows a 40× amplified photograph of conglomerates of agarose andglass spheres prepared according to Example 1(a);

FIG. 1B shows a 40× amplified photograph of selected sphericalconglomerates of agarose and glass spheres prepared according to Example1(a);

FIG. 1C shows a 40× amplified photograph of conglomerates of acrylicacid copolymer and single solid glass spheres prepared according toExample 11;

FIG. 2 illustrates a preferred embodiment of a fluid bed reactor; and

FIG. 3 illustrates another preferred embodiment of a fluid bed reactor;

FIGS. 4A and 4B show perspective sketches of another preferredembodiment of a down flow fluid bed reactor;

FIG. 5 illustrate the fluid bed particles of conglomerates according tothe invention in a down flow fluid bed reactor;

FIGS. 6A-6D illustrate cross-sections along the lines VIB, VIC, VID, VIEin FIG. 5; and

FIG. 7 illustrates the collection of fluid bed particles ofconglomerates according to the invention in a collecting vessel of aprotein purification batch process.

4. DETAILED DESCRIPTION

(a) Controlled Relative Density of Conglomerates

Within the present context the expression "relative density ofconglomerates" designates the density of the individual conglomerateparticles in the wet state, i.e. a state where the conglomerating agentis fully hydrated, but without any interstitial liquid betweenindividual conglomerate particles. This means that the liquid in whichthe conglomerate particles are used are determinant for the density ofthe conglomerate particles in as much as this liquid penetrates into thevolume of the conglomerating agent, solvates this and fill out thepores.

Further, the expression "relative density of the particles" designatesthe density of the particles relative to the density of the liquid inwhich the particles are to be used. This relative density is determinantfor the tendency of the particles to float or to sediment in a givenliquid. The relative density of conglomerate particles according to theinvention is thus dependent on the solvated density of theconglomerating agent, the concentration of conglomerating agent, thedensity of the basic particles (preferably impermeable to the liquid andsubstantially non-solvated) used to regulate the density and theconcentration of these.

The density of the solvated phase, i.e. the volume occupied by theconglomerating agent and the active substance will usually be dependenton the specific application of the particles and thus not allowable tobe regulated by variation of the concentration of conglomerating agent.

Therefore, according to the invention the density of the conglomerateparticles is regulated by the addition of basic particles having adensity free of choice with respect to the functionality of theconglomerate and also having a final concentration in the conglomeratefree of choice with respect to the functionality, i.e. the functionalityof the active principle within the volume of the conglomerating agent isnot disturbed by the density and concentration of the basic particles.

A crude estimate of the final density as a function of the concentrationof basic particles can be found by the following equation:

    Density of conglomerate=((d.sub.c ×v.sub.c)+(d.sub.b ×v.sub.b))/(v.sub.c +v.sub.b)

d_(c) =density of solvated conglomerating phase

d_(b) =density of basic particles

v_(c) =volume occupied by solvated conglomerating phase

v_(b) =volume occupied by basic particles.

Differences in the degree of salvation occuring in different solventshave to be corrected for. Thus, for certain conglomerating agents, e.g.heavily charged polymers for ionexchange chromatography, the degree ofsolvation, i.e. the volume of liquid taken up per gram dry weight, maydiffer with several hundred percent in fluids with different ionicstrength or pH.

By way of example the density of conglomerate particles comprisingagarose as the conglomerating agent and hollow glass spheres as basicparticles is regulated by the addition of hollow glass spheres to theliquified agarose, the amount added (for example measured as gram hollowglass spheres per ml agarose) being determinant for the density of thefinal conglomerate.

Assuming a density of the agarose phase to be 1.0 g/ml and the volumeused to be one liter (1000 ml) and the density of the hollow glassspheres to be 0.2 g/ml and the amount used to be 100 g (corresponding to500 ml) the calculated density would be:

    ((1.0×1000)+(0.2×500))/(1000+500)=0.73 g/ml

If only 50 g of hollow glass beads were added the calculated densitywould be:

    ((1.0×1000)+(0.2×250))/(1000+250)=0.84 g/ml

If instead of the hollow glass spheres, the basic particles used weresolid glass spheres with a density of 2.5 g/ml and 500 g were used tothe same amount of agarose, the calculated density would be:

    ((1.0×1000)+(2.5×200))/(1000+200)=1.25 g/ml

"Concentration of Basic Particles"

Generally, the basic particle concentration shall be as small aspossible in order to obtain as high a concentration of the activesubstance as possible. However, depending on the application, the basicparticles concentration by volume is selected from a group consistingof:

1-95%,

1.5-75%,

5-50%,

5-40%,

5-30%, most preferred.

"Dimensions of Conglomerates"

According to the invention, optimum dimensions of a conglomerate of thetypes according to the present invention will largely depend upon theuse to which they are to be put, although limitations dictated by thenature of the material and/or by the nature of the active substance andconglomerating agent within the conglomerate may also play a role.

From the point of view of achieving the greatest rate of interaction ofchemical species with a given mass of conglomerate of a particular type,it will generally be advantageous that the total surface area of theconglomerate is as large as possible, and thus that the size of theconglomerate is as small as possible.

In preferred aspects of a conglomerate according to the invention, thesize of substantially all of said conglomerates is within a rangeselected from the group consisting of:

1-10000 μm,

1-5000 μm,

1-4000 μm,

1-3000 μm,

1-2000 μm,

1-1000 μm,

50-500 μm.

The actual size preferred is dependent on the actual application and thedesired control of the dispersion properties, e.g. sedimentation andfloatation, of the conglomerate both properties being dependent on thedensity and the size of the conglomerate. Thus, for very fast separationflow rates conglomerates of relatively low or high densities andrelatively large sizes are preferred. However, large conglomerates maybe limited in diffusion in certain applications, e.g. when proteins haveto diffuse in and out of conglomerates and interact with activesubstances within the conglomerate.

Further, for conglomerates having the same density and size the, thediffusion properties of molecules within the conglomerate may depend onthe number of basic particles. Thus, for conglomerates having one basicparticle, the diffusion length may be shorter than for conglomerateshaving many smaller basic particles. In general, conglomerates of onlyone basic particle may be preferred when the molecular diffusion withinthe conglomerate is a limiting factor of the application.

Thus, for purification and binding of proteins and other high molecularweight substances which may diffuse slowly in the conglomerate, e.g. inthe conglomerating agent, the preferred size of conglomerates is withina range selected from the group consisting of:

1-2000 μm,

10-1000 μm,

50-750 μm,

100-500 μm, most prefered.

Further, particularly for purification and binding of proteins and otherhigh molecular weight substances in a batch process, the preferred sizeof conglomerate particles is within a range selected from the groups of:

1-2000 μm

250-2000 μm

500-2000 μm

500-1000 μm, most preferred.

Further, for enzyme reactions in which an enzyme immobilized within theinterior of the conglomerate reacts with a substrate of relatively lowmolecular weight, the prefered size of conglomerates is within rangesselected from a group consisting of:

10-10000 μm,

50-5000 μm,

100-3000 μm,

200-1000 μm, most preferred.

Especially for immobilisation of microorganims, the preferred size ofthe conglomerate is within ranges selected from the group consisting of:

0.5-50 mm

0.5-10 mm

0.5-5 mm, most preferred.

For a conglomerate within the context of the present invention to be ofuse, for example, in chromatographic separation processes, thetime-scale of the process of diffusion of fluid i.e. gaseous or liquidphases through the conglomerate, where relevant, should preferably beshort in order to ensure sufficiently rapid equilibration between extra-and intraparticular phases; this time-scale will often be of the orderof seconds.

(b) Basic Particles and Materials

In selecting basic particles for use as low or high density particlesaccording to the invention, the material of the particles depends on thepurpose. Generally, the material is to be sought among certain types ofnatural or synthetic organic polymers, primarily synthetic organicpolymers, inorganic substances and compounds, metallic elements, andalloys thereof, non-metallic elements, and gas bubbles.

"Synthetic Organic Polymers"

Among types of synthetic organic polymers which may possibly be ofinterest are resins of the phenol-formaldehyde type and ABS resins, butother classes of synthetic organic polymers, such as acrylic polymers,polyamides, polyimides, polyesters, polyethers, polymeric vinylcompounds, polyalkenes and substituted derivatives thereof, as well ascopolymers comprising more than one such said polymer functionality, andsubstituted derivatives of such copolymers, may well furnish suitablecandidates.

Particularly preferred low density basic particles are hollow plasticparticles.

"Inorganic Substances and Compounds"

However, from the point of view of cheapness and ready availability, insome cases it is advantageous to employ particles of inorganic material,especially since materials with the greatest mechanical rigidity aregenerally to be found amongst inorganic materials. Thus, material of thebasic particles employed in the conglomerate according to the inventionmay comprise a member selected from the group consisting of inorganicsubstances and compounds, metallic elements and alloys thereof, andnon-metallic elements.

In a preferred aspect, the material comprises a member selected from thegroup consisting of:

anhydrous forms of silicon dioxide, including amorphous silica andquartz;

metal silicates, including silicates of lithium, sodium, potassium,calcium, magnesium, aluminium and iron, and metal borosilicates, such asborosilicates of said metals, metal phosphates, includinghydroxyapatite, fluorapatite, phosphorite and autunite;

metal oxides and sulfides, including magnesium, aluminium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper and silveroxides;

non-metal oxides, including boric oxide;

metal salts, including barium sulfate;

metallic elements, including magnesium, aluminium, titanium, vanadium,chromium, manganese, iron, cobalt, nickel, indium, copper, silver, gold,palladium, platinum, ruthenium, osmium, rhodium and iridium, and alloysof metallic elements, such as alloys formed between said metallicelements;

crystalline and amorphous forms of carbon, including graphite, carbonblack and charcoal.

"Gas Bubbles"

Further, in a preferred aspect the material of the basic particles maycomprise gases such as air, nitrogen, carbon dioxide, or inert gases,e.g. He, Ne, Ar, Kr, and Xe, confined in a cavity.

"Siliceous Glassy or Ceramic Materials"

As mentioned earlier, the prior art discloses a number of examples ofhollow particles of siliceous glassy or ceramic material which may beused as low density hollow particles of conglomerates according to theinvention, these previously disclosed particles being obtainedrelatively cheaply and straightforwardly by deliberate synthesis or as afly-ash by-product of certain combustion processes.

Accordingly, in a further preferred aspect of the invention, thematerial of the basic particles employed in conglomerates of both lowand high density particles according to the invention is a glass,preferably an synthetic glass comprising silicon dioxide and/or asilicate.

In yet another preferred aspect of the invention, such material is asilicon dioxide-containing material derived from fly-ash, in which casethe material may be amorphous (e.g. glassy) or crystalline, or to someextent both amorphous and crystalline.

"Magnetic Materials"

For certain applications of a conglomerate, the material of the basicparticles may comprise an appropriate amount of magnetic material e.g.for confining or retaining the conglomerate within a particular regionof, for example, a process vessel or a chromatographic column, withoutthe need for the incorporation of physical means of confinement orretention, such as a filter.

Thus, a further aspect of the invention provides conglomerates of basicparticles of which the particles comprises a component selected from thegroup consisting of:

paramagnetic metallic elements, including iron, cobalt and nickel, andparamagnetic alloys, including alloys containing said paramagneticmetallic elements;

metal oxides, including iron(II) oxide, iron(III) oxide, cobalt(II)oxide and nickel(II) oxide;

metal salts, including cobalt(II) salts, e.g. cobalt(II) phosphate,chromium(III) salts, e.g. chromium(III) fluoride, and manganese(II)salts, e.g. manganese(II) carbonate.

"Basic Particle Structure"

Further, the material of the basic particles within the context of thepresent invention may be chemically and/or physically inhomogeneous. Forexample, it may have a layered structure involving one or more layers ofsimilar or different materials, e.g. various types of siliceousmaterials. Alternatively, for example, it may consist of a siliceousmaterial, such as a siliceous glassy material, containing particles orregions with a high content of a metal oxide or a metallic elementchemically reactive, e.g. as a catalyst.

(c) Active substances

Concerning the active substances to be introduced into the conglomerateaccording to the invention, this may, for example, be any type ofmaterial which is useful for a given application. Further, in one aspectof the invention the active substance may itself act as a conglomeratingagent keeping the basic particles together and providing mechanicalstability.

In another aspect of the invention the material of an active substancecomprises a member selected from the group consisting of organic andinorganic compounds or ions, metallic elements and alloys thereof,non-metallic elements, organic polymers of biological and syntheticorigin, membrane-enclosed structures, biological cells, and virusparticles.

In a preferred aspect, the active substance comprises a member selectedfrom the group consisting of:

ligands known per se in the field of chromatography, e.g. chargedspecies i.a. for ion exhange chromatography; proteins, dyes, enzymeinhibitors, specific ligands for specific proteins, e.g. biotin forpurification of avidin and other biotin binding proteins, carbohydratesfor purification of lectins or glycosidases, protein A, chelates, e.g.iminodiacetic acid; amino acids, e.g. arginine, lysine, and histidine;sulfated polymers including e.g. heparins; gelatins; benzhydroxamicacid; hydrophobic ligands, e.g. phenyl, hydrocarbons such as octylamine, octanol; thiophilic ligands, i.e. divinyl sulfone activatedsubstances coupled with mercaptoethanol or 4-hydroxy-pyridine,3-hydroxy-pyridine, 2-hydroxy-pyridine;

lipid vesicles;

microorganisms and enzyme systems;

virus particles, including attenuated and inactivated virus particles;

natural and synthetic polynucleotides and nucleic acids, including DNA,RNA, poly-A, poly-G, poly-U, poly-C and poly-T;

natural and synthetic polysaccharides and other carbohydrate basedpolymers, including agar, alginate, carrageenan, guar gum, gum arabic,gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum,agaroses, celluloses, pectins, mucins, dextrans, starches, heparins, andgelatins;

natural and synthetic peptides and polypeptides and other amino acidbased polymers, including albumins, hemoglobulins, immunoglobulinsincluding poly- and monoclonal antibodies, antigens, protein A, proteinG, lectins, glycoproteins such as ovomucoids, biotin binding proteinse.g. avidin and streptavidin, and enzymes e.g. proteases, and proteaseinhibitors;

synthetic organic polymers, including acrylic polymers, polyamides,polyimides, polyesters, polyethers, polymeric vinyl compounds,polyalkenes, and substituted derivatives thereof, as well as copolymerscomprising more than one such organic polymer functionality, andsubstituted derivatives of such copolymers;

food, medicals, and vaccines for fish and other animals living in water;

hydrated and anhydrous forms of silicon dioxide, including silica gel,amorphous silica and quartz;

metal silicates, including silicates of lithium, sodium, potassium,calcium, magnesium, aluminium and iron, and metal borosilicates,including borosilicates of said metals;

metal phosphates, including hydroxyapatite, fluorapatite, phosphoriteand autunite;

metal oxides, including magnesium, aluminium, titanium, vanadium,chromium, manganese, iron, cobalt, nickel, copper, and silver oxides,and paramagnetic metal oxides, including iron(II), iron(III), cobalt(II)and nickel(II) oxides;

metal salts, including barium sulfate, and paramagnetic metal salts,including combalt(II), chromium(III) and manganese(II) salts;

metallic elements, including magnesium, aluminium, titanium, vanadium,chromium, manganese, indium, copper, silver, gold, palladium, platinum,ruthenium, osmium, rhodium and iridium, and paramagnetic metallicelements, including iron, cobalt and nickel, and alloys of metallic andparamagnetic metallic elements, including alloys formed between saidmetallic and paramagnetic metallic elements.

"Introduction of Active Substance into Conglomerates"

Generally, the active substance may be introduced into the conglomeratein a number of ways depending on the nature of the active substance,conglomerating agent, and the conglomerate itself, e.g. its pore size.Thus, both low and high molecular weight ligands may be incorporatedduring conglomeration either by entrapment or by chemical cross-linkingor by co-polymerisation. Further, both low and high molecular weightligands may be chemically coupled to a conglomerating agent before orafter conglomeration, or they may be coupled to precursor monomers orpolymers introduced together with the conglomerating agent during theconglomeration provided the desired functions of the active substance iskept intact or may be reestablished before use. However, if the means ofconglomerating damages or destroys the functioning of the activesubstance, the fragile active substance may be introduced afterconglomeration provided the conglomerate has been designed with suitablepore sizes to allow access to its interior.

"Introduction via Liquid Media"

Materials within several of the above categories, for example lipidvesicles, virus particles, certain polypeptides, and certain metalsilicates and other metal salts, may be introduced into the conglomeratein the form of solutions, suspensions or dispersions in suitable liquidmedia.

"In Situ Formation/Sequential Introduction"

Materials within some of the other categories, for example numerouspolymers and copolymers, certain metal phosphates, certain metal oxides,e.g. silver oxide, and certain metallic elements, e.g. silver, may beintroduced into the conglomerate by forming them in situ via a series ofsteps involving the sequential introduction into the conglomerate of aseries of reagent solutions; for example silver oxide might be depositedin situ within conglomerate by partially infusing them with an aqueoussolution of a soluble silver(I) salt, e.g. silver(I) nitrate, and thenpartially infusing them with an aqueous solution of a base, e.g. sodiumhydroxide. The liquid solvent(s) may then, if desired, be removed fromthe conglomerate by, e.g., subjecting the conglomerate to a vacuumtreatment.

"In Situ Formation/Thermal Treatment"

In some cases it may be possible to form an active substance in situwithin the conglomerate by thermal treatment of a substance which hasinitially been introduced into or formed within the conglomerate via theintroduction of one or more solutions, suspensions or dispersions inliquid media, although this obviously will require that the material ofthe conglomerate itself and the conglomerating agent suffer nodetrimental effects as a result of the thermal treatment; for example,it is well known that the noble metals platinum and rhodium can beformed in finely divided, highly catalytically active form by heatingalmost any complex or binary compounds of the elements, e.g. (NH₄)₂PtCl₆ ! or (NH₄)₃ RhCl₆ !, at temperatures above about 200° C. in thepresence of oxygen or air.

"Incorporation of Organic Polymers or Copolymers; In SituPolymerisation"

For the use of conglomerates according to the invention in variouschromatographic procedures, e.g. ion exchange chromatography, and inother procedures, for example solid-phase peptide synthesis, theconglomerates in question may incorporate organic polymers orcopolymers. By way of example, the application of a permeableconglomerate according to the invention to peptide synthesis employingthe classical chemical methodology of Merrifield see, e.g., Barany etal, Int. J. Peptide Protein Res. 30 (1987) pp. 705-739! will initiallyrequire the in situ formation of a cross-linked styrene/divinylbenzenecopolymer resin by polymerization of styrene monomer containing,typically, about 1-2% of divinylbenzene; the resin may then befunctionalized by subsequent treatment of the resin-containingconglomerate with solutions of the appropriate reagents.

Thus, in a further aspect of a conglomerate according to the invention,the active substance comprises a polymer or a copolymer formed in situwithin the conglomerate by a procedure comprising the steps of:

immersing the conglomerate in a solution, in a liquid solvent or solventmixture, of one or more components which can polymerize or copolymerizeto form a polymer or a copolymer or mixtures thereof, the solutionoptionally containing a polymerization catalyst or initiator,

allowing the solution to at least partly fill the conglomerate via thethrough-going pores,

allowing the polymer-/copolymer-forming components topolymerize/copolymerize to form solid polymer(s)/copolymer(s) therein,

optionally substantially removing any liquid solution remaining withinthe conglomerate,

optionally further treating the polymer-/copolymercontainingconglomerates as to:

(i) at least partly chemically derivatize and/or modify thepolymer(s)/copolymer(s) within the conglomerate and/or

(ii) introduce further components into the conglomerate.

"Incorporation of Fragile Active Substances"

Materials other than those mentioned above may also conceivably beincorporated as components of porous conglomerates; for example, forcertain biotechnological applications, such as the preparation ofvaccines, antibodies or toxins, or cell cultivation for the productionof metabolites (e.g. the production of ethanol by yeast cells) it may bedesirable, according to the invention, to introduce live or dead cellsof human, animal, plant, fungal or microoganism origin, or organelles(such as nuclei, mitochondria, chloroplasts or lysozomes) of similarorigin, into conglomerates in situ. This will, of course, necessitatethe provision of relatively large permeable conglomerates havingthrough-going pores of a suitably large size, e.g. of the order of ca.5-20 μm in the case of several types of human cells such as it will thenoften be necessary or desirable, after the introduction of such cells ororganelles, to coat the resulting conglomerates by a suitable treatment,so as to retain the cells or organelles within the conglomerates butallow migration of smaller species into or out of the conglomerates.This may be done by coating the conglomerates with a suitable membranematerial having a suitable permeability.

"Pore Sizes and Their Formation"

The optimum size or size-range of the through-going pores will, ofcourse, vary very considerably, depending on the use to which thepermeable conglomerate is to be put. Such pore sizes are difficult tocharacterize quantitatively; however, in terms of the size of themolecules which are to be capable of passing through the pores, arealistic upper exclusion limit for macromolecules, notably biologicalmacromolecules, such as proteins, will often be a molecular weight ofthe order of magnitude of 10⁸. The practical lower limit for pore sizewill generally be set by physico-chemical considerations, e.g. thedetailed chemical structure of the outer part and the manner in whichthe outer part material dissolves or reacts during the pore-formationprocess. Although possibly rather difficult to achieve, the formation ofthrough-going pores with sizes of the order of a few Angstrom would beadvantageous, in that the resulting permeable conglomerate in questionwould be expected to be applicable as so-called "molecular sieves"; forexample, a typical application of permeable conglomerates with pores ofthis size would be as materials for removing traces of water fromorganic solvents, and the relatively large internal cavity volume ofsuch conglomerates should confer a large drying capacity per per volumeunit of conglomerate.

Pore sizes may typically be formed by methods known per se, e.g. bysimply controlling the concentration of the conglomerating agent. Thus,for agarose or acrylamide derivatives a larger concentration willprovide a smaller pore size. However, other methods may be applieddepending on the conglomerating agent and e.g. the incorporated polymersand copolymers.

"Activation or Derivatization"

In cases where the conglomerating agent may not have the properties tofunction as an active substance, the conglomerating agent, or agents, orpolymers introduced in the conglomerate, may be derivatized to functionas one or more active substances by procedures of activation orderivatisation well known per se. Thus, materials comprising hydroxyl,amino, amide, carboxyl or thiol groups may be activated or derivatizedusing various activating chemicals, e.g. chemicals such as cyanogenbromide, divinyl sulfone, epichlorohydrine, bisepoxyranes,dibromopropanol, glutaric dialdehyde, carbodiimides, anhydrides,hydrazines, periodates, benzoquinones, triazines, tosylates, tresylates,and diazonium ions.

(d) Conglomerating Agents

In selecting the conglomerating agent for use as a means of keeping thebasic particles together and as a means for binding, entrapping, orcarrying the active substance, the conglomerating material is to besought among certain types of natural or synthetic organic polymers, andinorganic substances.

"Organic Polymers"

In one aspect of the invention the material of the conglomerating agentcomprises a member selected from the group consisting of organicmonomers and polymers of biological and synthetic origin.

In a preferred aspect, the conglomerating agent comprises a memberselected from the group consisting of:

natural and synthetic polysaccharides and other carbohydrate basedpolymers, including agar, alginate, carrageenan, guar gum, gum arabic,gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum,agaroses, celluloses, pectins, mucins, dextrans, starches, heparins, andgelatins;

synthetic organic monomers and polymers resulting in polymers, includingacrylic polymers, polyamides, polyimides, polyesters, polyethers,polymeric vinyl compounds, polyalkenes, and substituted derivativesthereof, as well as copolymers comprising more than one such organicpolymer functionality, and substituted derivatives thereof.

"Inorganic Substances"

In another preferred aspect, the conglomerating agent comprises a memberselected from the group consisting of:

hydrated and anhydrous forms of silicon dioxide, including silica gel,amorphous silica and quartz;

metal silicates, including silicates of lithium, sodium, potassium,calcium, magnesium, aluminium and iron, and metal borosilicates,including borosilicates of said metals;

metal phosphates, including hydroxyapatite, fluorapatite, phosphoriteand autunite;

metal oxides and sulfides, including magnesium, aluminium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, and silveroxides, and paramagnetic metal oxides, including iron(II), iron(III),cobalt(II) and nickel(II) oxides;

metal salts, including barium sulfate, and paramagnetic metal salts,including cobalt(II), chromium(III) and manganese(II) salts;

metallic elements, including magnesium, aluminium, titanium, vanadium,chromium, manganese, indium, copper, silver, gold, palladium, platinum,ruthenium, osmium, rhodium and iridium, and paramagnetic metallicelements, including iron, cobalt and nickel, and alloys of metallic andparamagnetic metallic elements, including alloys formed between saidmetallic and paramagnetic metallic elements.

"The Active Substance as Conglomerating Agent"

In one aspect of the invention the conglomerating agent may be omittedin the sense that the active substance itself can function as aconglomerating agent. Thus, as mentioned, in a preferred aspect of theinvention, the active substance may e.g. function as a conglomeratingagent. In this case the conglomerating agent may comprise a memberselected from the group consisting of:

microorganisms and enzyme systems;

natural and synthetic polynucleotides and nucleic acids, including DNA,RNA, poly-A, poly-G, poly-U, poly-C and poly-T;

natural and synthetic polysaccharides and other carbohydrate basedpolymers, including agar, alginate, carrageenan, guar gum, gum arabic,gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum,agaroses, celluloses, pectins, mucins, dextrans, starches, heparins, andgelatins;

natural and synthetic peptides and polypeptides and other amino acidbased polymers, including albumins, hemoglobulins, immunoglobulinsincluding poly- and mono clonal antibodies, antigenes, protein A,protein G, lectins, glycoproteins such as ovomucoids, biotin bindingproteins e.g. avidin and streptavidin, and enzymes e.g. proteases, andprotease inhibitors;

special synthetic organic polymers, including specifically designedacrylic polymers, polyamides, polyimides, polyesters, polyethers,polymeric vinyl compounds, polyalkenes, and substituted derivativesthereof, as well as copolymers comprising more than one such organicpolymer functionality, and substituted derivatives thereof;

special hydrated and anhydrous forms of silicon dioxide, includingspecifically design silica gel, amorphous silica and quartz;

special metal silicates, including specifically designed silicates oflithium, sodium, potassium, calcium, magnesium, aluminium and iron, andmetal borosilicates, including borosilicates of said metals;

special metal phosphates, including specifically designedhydroxyapatite, fluorapatite, phosphorite and autunite;

special metal oxides sulfides, including specifically designedmagnesium, aluminium, titanium, vanadium, chromium, manganese, iron,cobalt, nickel, copper, and silver oxides, and paramagnetic metaloxides, including iron(II), iron(III), cobalt(II) and nickel(II) oxides;

special metal salts, including specifically designed barium sulfate, andparamagnetic metal salts, including combalt(II), chromium(III) andmanganese(II) salts;

special metallic elements, including specifically designed magnesium,aluminium, titanium, vanadium, chromium, manganese, indium, copper,silver, gold, palladium, platinum, ruthenium, osmium, rhodium andiridium, and paramagnetic metallic elements, including iron, cobalt andnickel, and alloys of metallic and paramagnetic metallic elements,including alloys formed between said metallic and paramagnetic metallicelements; and

other materials used as active substance provided they can conglomeratethe basic particles.

"Activation or Derivatization of Conglomerating Agents"

However, in cases where the conglomerating agent may not have theproperties to function as an active substance, the conglomerating agentmay be derivatized to function as one or more active substances byprocedures of activation or derivatisation well known per se. Thus,materials comprising hydroxyl, amino, amide, carboxyl or thiol groupsmay be activated or derivatized using various activating chemicals, e.g.chemicals such as cyanogen bromide, divinyl sulfone, epichlorohydrine,bisepoxyranes, dibromopropanol, glutaric dialdehyde, carbodiimides,anhydrides, hydrazines, periodates, benzoquinones, triazines, tosylates,tresylates, and diazonium ions.

(e) Illustration of Conglomerate Particles

FIG. 1A shows a 40× amplified photograph of aspherical conglomerateparticles 10 having 1-2 mm of diameter and being prepared by distibutingunicellar glass microspheres 11 in conglomerating agarose 12 accordingto Example 1(a).

FIG. 1B shows a 40× amplified photograph of selected sphericalconglomerate particles also prepared according to Example 1(a).

FIG. 1C shows a 40× amplified photograph of aspherical conglomeratedparticles 13 comprising a single solid glass sphere 14 and an acrylicacid copolymer 15 prepared according to Example 11.

(f) Fluid Bed Reactors

"C Reactor"

FIG. 2 illustrates a cross section of a preferred embodiment of a fluidbed reactor 20 composed of a outer cylinder 21, a top lid 22 with inlet221 and connection for a stirrer 222, and a bottom lid 23 with outlet231. Further, an inner cylinder 24 having holes and mounted on a support25 attached to support blocks 251 and 252 allowing passage of the fluid.Stirring is performed at a suitable rate of rotation within the innercylinder 24 to assure a sharp lower boundary 26 of the fluid bedconglomerates. Without stirring, the bed of light conglomerates floatagainst the top lid 22 and have a lower boundary 27.

"T reactor"

FIG. 3 illustrates a cross section of another preferred embodiment of afluid bed reactor 30 similar to the reactor illustrated in FIG. 2 exceptthat the inner cylinder 24 is replaced by an inverse funnel 34 having anupper outlet 341 and supported by a support cylinder 35 that is openupwards. Conglomerates arriving below the funnel in the turbulens freevolume will rise up through the upper outlet 341 while fluid flow downthrough the outlet 231.

The stirrer is placed right below the surface 36 and stirring isperformed at a suitable rate of rotation to provide a sharp lowerboundary 37 of the conglomerates.

Without stirring, the bed of light conglomerates have a lower boundary38.

"Controlled Fluid Distribution in Fluid Bed Reactors"

FIGS. 4A and 4B show perspective sketches of a preferred embodiment of adown flow fluid bed reactor 40.

A dc-motor 41 controlled by a variable speed control 42 providesrevolutions of a stirrer 43, which in a mixing zone A agitates the fluidbed particles to generate a turbulent flow of the fluid flowingdown-ward.

A sharp interface (generally of few particle diameters) is reached atthe non-mixing zone B in which the particles are stationary and an evenand smooth distribution of the fluid is obtained.

In order to adapt the conditions of agitation the length of the fluidbed column can be changed by means of interchangeable chromatographictubes 45.

(A) "Down-Flow Fluid Bed Reactor"

FIG. 5 shows a longitudinal section of a segment of a down-flow fluidbed reactor 50 comprising a vertical cylinder 54 and a fluid bed A,B,Cof particles 51,52,53 suspended in a down-flow fluidizing fluid 56 letin through an inlet at the top of the reactor vessel, the particles51,52,53 having a specific gravity less than that of the fluid. A gashead 57 is above the surface along the line VIA--VIA.

The upper part of the fluid bed is agitated by a plate formed mechanicalstirrer 55 dividing the bed into a mixing zone A, a non-mixing zone B,and an exit zone C.

In the mixing zone A, the agitated fluid bed particles 51 movedynamically which generates a turbulent flow of the fluid. Theturbulence decreases down the mixing zone A. A sharp interface VIC--VICis reached at the non-mixing zone B in which the particles 52 are in astationary fluidized state. Across the interface VIC--VIC, the fluidflow is distributed evenly, and a smooth fluid flow is obtained in thenon-mixing zone B.

In the exit zone C, the pooled reacted and/or unreacted fluid 57 leavethe fluid bed at an interface VID--VID, where particles 53 can becomeseparated from the fluid bed by the fluid flow.

FIGS. 6A-6C show cross sections of the mixing zone A along the linesVIB--VIB, VIC--VIC, and VID--VID, respectively, of the FIG. 5. Thus,FIG. 6A shows a cross section of essentially randomly moving particles51, and FIGS. 6B & 6C show cross sections of essentially stationaryfluidized particles 52 and 53.

FIG. 6D shows a cross section, along the line VIE--VIE, essentiallywithout particles.

(g) Batch Reactors

In solid phase reactions, e.g. in adsorption of at least one selectedsubstance from a fluid medium or in an enzyme reaction procedure, thereaction may be performed in a batch reactor. Generally the procedurewill be to bring the conglomerate particles in contact with the liquidto be treated in a stirred tank for a certain time (the length of whichis determined by the rate of adsorption of the conglomerate particles orthe rate of enzymatic reaction, respectively) followed by separation ofthe conglomerate particles from the liquid.

FIG. 7 shows a collection vessel applied in a protein purification batchprocess. Conglomerate particles previously contacted with the adsorbentor reagent in the liquid to be treated are pumped through the inletvalve 71 of the collection vessel 70. The low density conglomerateparticles raise continuously to the top 75 of the vessel as they enterthrough a pipe 72 and are then trapped in the vessel (the valve 73 beingclosed), while the extract is leaving the vessel through the outletvalve 74.

After collection of the conglomerate particles in the top section 75 ofthe vessel, the vessel and the particles are washed by pumping asuitable washing liquid e.g a solution of sodium chloride through thenow opened valve 73 in the top and out through the outlet valve 74 inthe bottom of the vessel (the valve 71 being closed).

Particularly for purification and adsorption purposes, the boundadsorbent is then eluted from the conglomerate particles (still being inthe top of the vessel) by pumping a suitable eluent e.g. hydrochloricacid into the vessel through valve 74 (arrow in parenthesis) in thebottom, up through the bed of conglomerate particles and out throughvalve 73 in the top of the vessel 75 (the valve 71 being closed and theparticles being retained within the vessel by the coarse filter 76).

5. EXAMPLES

All solutions employed in the following examples are aqueous solutionsunless otherwise indicated.

Example 1

Preparation of conglomerates based on 3M's unicellarglass microspheres"Glass Bubbles", B28/750, C15/250, and E22/400, soda-lime-borosilicate!having a mean density of 0.28 g/cm³, 0.15 g/cm³, and 0.22 g/cm³,respectively.

(a) Low Density Agarose Hollow Glass Spheres Conglomerated Particles

300 ml soya bean oil was heated together with 3 ml sorbitane sesquiolateto 60° C. 5 ml 6% agarose (HSA, Litex) in water was heated and 0.5 ghollow glass spheres (3 M, B28/750) having a mean density of 0.28 g/cm³were added under stirring. Following mixing of the agarose and glassmicrospheres the suspension was added to the soya bean oil under heavilystirring. The emulsion formed was stirred at about 60° C. in fiveminutes and cooled to 20° C. The solidified agarose particles containingbasic particles of hollow glass spheres were washed on a sintred glassfilter with sufficient ether until all soya bean oil was removed. Theconglomerate was then washed with water. The conglomerate had a lowdensity and was floating on water.

(b) Low Density Agarose Hollow Glass Sphere Conglomerated Block PolymerParticles

300 ml 4% agarose was prepared by heating 12 g agarose (HSA, Litex) in300 ml water. 9 g hollow glass spheres (C15/250, 3M) was added and themixture was stirred until a homogeneous suspension was obtained. Thesuspension was cooled to 60° C. under steadily stirring and the fluidsuspension poured on to an efficiently cooled surface. The agarose glasssphere suspension was gelated over a short period. The gel block had ahomogeneous distributed content of hollow glass spheres. After coolingthe gel block was blended and the granulate was sorted according to sizeand flow ability by means of "reverse sedimentation".

(c) Low Density Polyamide Hollow Class Sphere Conglomerated Particles

5 g acrylamide and 0.5 g N,N'-methylenbis(acrylamide) were dissolved in100 ml 0.1 M potassiumhydrogenphosphate-HCl, pH 7.0. 3 g hollow glassspheres (C15/250, 3M) were added under stirring. Following the formationof a homogeneous suspension a catalyst of 1 g ammoniumpersulphate and0.5 ml N,N,N',N'-tetramethylethylendiamine was added for thepolymerisation. Stirring was continued until a highly viscous suspensionwas formed. Following polymerisation the polymer block containing hollowglass spheres was blended as described under (b).

(d) Low Density Gelatin Hollow Glass Sphere Conglomerated Particles

Five samples of 100 ml 5% gelatin (35° C.) in 0.15 M sodium chloridewere added hollow glass spheres (E22/400, 3M) in increasing amounts:

A: 0 g

B: 2 g

C: 5 g

D: 20 g

E: 27 g

After adjustment of pH to 5.5 all samples were added 2.0 ml glutaricdialdehyde (25% solution, cat.no.: 820603, Merck) under thoroughstirring. After 24 hours of incubation at room temperature thepolymerized matrices were disintegrated in a blender. The resultingparticles were separated from fines by reverse sedimentation (for A bysedimention as these particles were not floating). The particles werethen collected on a glass filter and drained for excess water by vacuumsuction on the glass filter. The wet but drained particles were thenweighed and the particle volume determined by adding a known amount ofliquid followed by determination of the total volume. The followingparticle densities were obtained:

    ______________________________________    Measured Density                   Calculated Density    ______________________________________    A:        1.0 g/ml 1.00 g/ml    B:        0.9 g/ml 0.93 g/ml    C:        0.8 g/ml 0.85 g/ml    D:        0.6 g/ml 0.63 g/ml    E:        0.5 g/ml 0.57 g/ml    ______________________________________

(e) Low Density Gelatin Hollow Glass Sphere Conglomerated Particles, andImmobilization of Horse-Radish Peroxidase

1 g of horse-radish peroxidase (grade II, Kem-En-Tec, Denmark) wasdissolved in a solution of 100 ml 10% gelatin (cat.no.: G-2500, Sigma)and 0.5 M sodium chloride (35° C.). 10 g hollow glass spheres (B28/750,3M) were added under stirring. After adjustment of pH to 5.5, 2 mlglutaric dialdehyde (25% solution, cat.no.: 820603, Merck) was addedwith thorough stirring. The resulting gel was incubated at roomtemperature for 2 hours and then disintegrated in a blender. Thefloating particles were separated from fines and non-floating particlesby inverse sedimentation. The yield of wet, packed particles was approx.120 ml. The size range was determined to be from about 200 to about 500μm in diameter.

(f) Low Density Gelatin Glass Sphere Conglomerated Particles, andImmobilization of Yeast Cells

50 g bakers yeast cells were suspended in a solution of 100 ml 10%gelatin and 0.15 M sodium chloride at 35° C. The suspension was added 20g hollow glass spheres (B28/750, 3M). After adjustment of pH to 5.5, 2ml glutaric dialdehyde (25% solution, cat.no.: 820603, Merck) was addedwith stirring. After two hours of incubation at room temperature theresulting block polymer was disintegrated in a blender and the particleswere washed with 5 liters of 0.15 M sodium chloride. Non-floatingparticles were separated from floating particles by inversesedimentation. Approximately 200 ml of packed floating particlescontaining yeast cells were obtained. The size of these particlesspanned from about 150 to about 750 μm.

The ability of the floating immobilized yeast cells to ferment glucosewas evident from the evolvement of carbondioxide, when incubated in a10% glucose solution.

(g) Low Density Gelatin Glass Sphere Conglomerated Particles, andImmobilization of Yeast Cells

50 g bakers yeast cells were suspended in 100 ml 10% gelatin (cat.no.:G-2500, Sigma), 0.15 M sodium chloride at 35° C. The suspension wasadded 20 g hollow glass spheres (B28/750, 3M). After thorough mixing thesuspension was cooled by pouring onto an ice cold glassplate making thesuspension into a firm gel. The resulting gel was disintegrated in ablender and the particles washed with 5 liters of 0.15 M sodiumchloride. Non-floating particles were separated from floating particlesby inverse sedimentation. Approximately 200 ml of packed floatingparticles containing yeast cells were obtained. The size of theseparticles spanned from about 150 to about 750 micrometers. The abilityof the floating immobilized yeast cells to ferment glucose was evidentfrom the evolvement of carbondioxide, when incubated in a 10% glucosesolution.

(h) Low Density Agar-Gelatin Glass Sphere Conglomerated Particles I

2 g agar (Bacto-agar, Difco), and 3 g gelatin (cat.no.: G2500, Sigma)was dissolved in 100 ml 0.15 M sodium chloride by brief heating to theboiling point. After cooling to about 56° C. 10 g hollow glass beads(B28/750, 3M) were added. pH was adjusted to 4.0 with 5 M acetic acidfollowed by the addition of 2 ml glutaric dialdehyde (25% solution, Cat.No. 820603, Merck) with thorough stirring. The resulting polymer blockwas cooled to room temperature and incubated for 24 hours followed bydisintegration in a blender.

Floating particles were separated from fines and non-floating particlesby inverse sedimentation followed by collection of the floatingparticles on a glass filter. The yield of floating conglomerateparticles was 95 ml packed wet particles.

(i) Low Density Agar-Gelatin Glass Sphere Conglomerated Particles II

2 g agar (Bacto-agar, Gibco) and 3 g gelatin (cat.no.: G-2500, Sigma)was dissolved in 100 ml 0.15 M sodium chloride by brief heating to theboiling point. After cooling to about 56° C. 10 g hollow glass beads(B28/750, 3M) were added. The suspension was then cooled by pouring itonto an ice-cold glassplate. The resulting gel block was incubated for24 hours at 4° C. followed by disintegration by blending in ice-water.The conglomerate floating gel-particles were separated from non-floatingparticles by inverse sedimentation and then collected on a glass filter.The yield was 105 ml of packed, wet particles.

The particles were then suspended in 200 ml 0.1 M potassium phosphatebuffer pH 6.5 and crosslinked for two hours by addition of 10 mlglutaric dialdehyde (25% solution, 820603, Merck).

(j) Low Density Chitosan Glass Sphere Conglomerated Particles

A 4% solution of chitosan (Cat. No.: 22741, Fluka) was prepared byheating 12 g chitosan in 300 ml 10% v/v acetic acid. The viscoussolution was cooled to about 40° C. followed by addition of 20 g hollowglass beads (B28/750, 3M). 3 ml glutaric dialdehyde was added (25%solution, 820603, Merck) with thorough stirring. The resulting polymerblock was incubated for 24 hours at room temperature followed bydisintegration in a blender.

The conglomerate floating gel-particles were separated from non-floatingparticles by inverse sedimentation in 0.1 M sodium chloride and thencollected on a glass filter. The yield was 400 ml of packed, wetparticles with a diameter from about 200 μm to about 800 μm.

(k) Vinyltriethoxysilan Coated Glass Spheres and Polyamide ConglomeratedParticles

(A) "coating of the glass spheres"

75 g (dry) hollow glass spheres (C15/250, 3M) were mixed with 500 ml 1%vinyltriethoxysilan solution in 0.1 M acetic acid and the suspension wasstirred for one hour. The vinyltriethoxysilan solution was removed byfiltration on a glass filter.

(B) "Conglomerating acrylamide and glass spheres"

1.5 g N,N'-methylenbisacrylamide was dissolved in 10 ml ethanol andmixed with 8.5 g acrylamide dissolved in 90 ml water. 15 gvinyltriethoxysilan coated glass spheres, from (A), was added understirring. 0.5 g ammoniumpersulphate and 0.5 mlN,N,N',N'-tetramethylethylendiamine was added as polymerisationcatalysts after a homogeneous suspension was reached. The stirring wascontinued until the polymer block was formed. The polymer block wassubsequently blended as described in Example 1(b) and "fines" wereremoved by "inverse sedimentation". This procedure resulted in approx.100 ml low density conglomerate.

Example 2

Chemical Derivatisation of Low Density Agarose Glass SphereConglomerated Particles

10 g (dried, wet weight) agarose conglomerate spheres containing hollowglass spheres from Example 1 were suspended in 100 ml 0.5 Mpotassiumphosphate/sodiumhydroxide pH 11.4. 10 ml divinyl sulfon og 50mg sodiumborohydrid were added under stirring. The suspension wasstirred at room temperature for three hours and the spheres were washedwith water on a glass filter. The spheres were then activated chemically(i.e. a method out of many possibilities) and were ready for coupling ofother substances. As an example mercaptoethanol was coupled forsalt-dependent chromatopgraphy: The spheres were reacted with 5%mercaptoethanol in water that had been titrated to pH 9.5 with 1 Msodiumhydroxide for 3 hours at room temperature.

The spheres were then washed thoroughly with destined water and wereready to use in purification of proteins using salt-dependentchromatopgraphy.

Example 3

Purification of Human Immunoglobulin from Untreated Blood

100 g (dried, wet weight) divinylsulfon and mercaptoethanol treatedagarose conglomerate spheres equilibrated with and suspended in 50 ml0.75 M ammoniumsulphate were placed in a cylindrical glass column withan inner diameter of 5 cm and length of 10 cm. The glass column wassealed at the top and bottom using unscrewing plastic caps. The bottomlit had an outlet with a tube piping in the middle while the top lit hada corresponding inlet and a mechanical stirrer. The mechanical stirrerprovides stirring through a air tight collar for stirring theconglomerate spheres contained in the column. The stirring propeller wasdesigned to avoid fluid flow that carries the agarose conglomeratespheres down to the outlet in the bottom column. 2 l unfiltrated and notcentrifugated human blood (i.e. outdated blood from a blood bank) havingbeen added ammoniumsulphate to a final concentration of 0.75 M is leadthrough the column from the top with a flow of 10 ml/min under stirringwith the abovementioned stirrer (i.e. to avoid the formation of channelsthrough the fluid bed). 2000 ml of 0.75 M ammoniumsulphate was added atthe same flow rate for washing non-bound proteins and particulates.Finally, the bound proteins were eluted from the conglomerate spheres byleading 500 ml of 0.1 M sodium chloride through the column.

About 5 g human immunoglobulin was eluted in the sodium chloridefraction. Qualitative analysis showed a high purity of immunoglobulinhaving a very small contamination of albumin (<1%).

A corresponding purification of immunoglobulins with divinylsulfon andmercaptoethanol treated agarose spheres without hollow glass spheres wasnot possible in a traditionally packed column because of clogging of thecolumn by the red blood cells and other sticky materials in bloodplasma.

Example 4

Immunosorption

Agarose conglomerate spheres containing 4% agarose and produced asdescribed in Example 1 were activated with divinylsulfon as described inExample 2.

10 g (drained, wet weight) activated gel was coupled to rabbitimmunoglobulin by incubation of the gel over night with 20 ml rabbitimmunoglobulin solution (10 mg immunoglobulin/ml in 0.1 Msodiumhydrogencarbonate/sodiumhydroxide buffer, pH 8.6 and 5% w/vpolyethylenglycol MW 20,000). Excess active groups were blocked byincubation of the gel with 0.5 M ethanolamine/HCl, pH 9.0 for threehours. The gel was coupled with more than 80% of the added rabbitimmunoglobulin.

The floating conglomerate spheres having rabbit immunoglobulin attachedcould then be applied in an apparatus corresponding to the one inExample 3 for adsorption of antibodies against rabbit immunoglobulinfrom untreated serum of previously pure rabbit immunoglobulin immunizedgoats. The separated antibody was of a purity and activity correspondingto that obtained with conventionally packed columns using filtered andcentrifuged antiserum.

Example 5

Preparation of Ion Exchange Conglomerates.

(a) Cation Exchange Conglomerates. Conglomeration of polyacrylicacid/acrylamide/N,N'-methylen-bis(acrylamide) and hollow glass spheres.

300 ml destined water was added to 25 ml acrylic acid, 100 ml ethanol,10 g N,N'-methylen-bis(acrylamide), 25 g acrylamide, 2 gammoniumpersulphate, 25 g hollow glass spheres (B28/750, 3M) and 2 mlN,N,N',N'-tetramethylethylendiamine. The mixture was stirred until ahomogeneous suspension was achieved and then titrated to pH 8.5 with 5 Msodiumhydroxide under steadily stirring. Stirring was continued untilpolymerisation of the suspension ocurred. Following polymerisation theblock was blended as described in Example 1(b) and "fines" wereseparated by means of "inverse sedimentation". Following a thoroughlywash of the particles with water, 0.1 M HCl and 0.1 M NaCl, the contentof carboxyl groups in the gel was determined to be about 250 μmol per gdrained wet gel by simple titration.

(b) Conglomeration of Acrylic Acid/acrylamide/N,N'-methylenbisacrylamideand Vinyltriethoxysilan Coated Hollow Glass Spheres

60 g (dry) hollow glass spheres (C15/250, 3M), 40 ml acrylic acid, 32 gacrylamide, 8 g N,N'-methylenbisacrylamide and 5 ml vinyltriethoxysilanewas added to 300 ml distilled water. The mixture was stirred for onehour and brought to pH 7 with cold 27.4% sodium hydroxide. 1 gammoniumpersulphate and 1 ml N,N,N',N'-tetramethylethylendiamine wasadded as polymerisation catalysts and stirring was continued until thepolymer block was formed. The polymer block was subsequently blended asdescribed in Example 1(b) and "fines" were removed by "inversesedimentation". In a batch-protein binding assay, pH 9, 50 mM TRIS/HCl,1 g of drained wet conglomerate was able to bind 96% of 190 mg offeredlysozyme.

(c) Conglomeration of AcrylicAcid/methacrylamide/N,N'-methylenbisacrylamide and VinyltriethoxysilanCoated Hollow Glass Spheres

Following the procedure described in Example 5(b), this ion exchangeconglomerate was prepared as by using methacrylamide in exchange foracrylamide. In a batch protein binding assay, pH 9, 50 mM TRIS/HCl, 1 gof the resulting drained wet conglomerate was able to bind 92% of 190 mgoffered lysozyme.

(d) Conglomeration of AcrylicAcid/methacrylamide/N,N'-methylenbisacrylamide and VinyltriethoxysilanCoated Hollow Glass Spheres

Following the procedure described in Example 5(c), this ion exchangeconglomerate was prepared as by using only 20 ml acrylic acid, 16 gmethacrylamide and 4 g N,N'-methylenbisacrylamide, giving theconglomerate a lower dry weight content allowing larger proteins todiffuse in and out of the conglomerate. In a batch protein bindingassay, pH 9, 50 mM TRIS/HCl, 1 g of conglomerate was able to bind 92% of190 mg offered lysozyme.

Example 6

Immobilised Enzyme. Immobilization of glucose oxidase.

10 g divinylsulfon activated agarose conglomerate spheres from Example 2were mixed with 20 ml of a solution of glucose oxidase from Aspergillusniger (10 mg/ml in 1 M potassiumhydrogenphosphate/sodiumhydroxidebuffer, pH 10.5). The mixture was left for three hours and the uncoupledglucose oxidase was washed out of the spheres by 1 M sodium chloride.

The enzyme coupled conglomerate spheres showed glucose oxydase activitywith glucose as a substrate. The development of hydrogen peroxide wasdetected as a brown colouring of the gel and solution by coupling thereaction with peroxidase (horse-radish peroxidase) oxidation oforthophenylen diamine.

Example 7

Immobilization of N-acetylglucosamine for the Separation of Wheat GermAgglutinin.

Conglomerate spheres containing 4% agarose and produced as described inExample 1(b) were activated with divinyl sulfon as described in Example2. 10 g (dried, wet weight) of the activated gel was coupled toN-acetylglucosamine by incubating the gel over night with 20 ml 0.5 Mpotassiumphosphate/sodiumhydroxide buffer pH 11.5 containing 50 mgN-acetylglycosamine per ml. Following incubation the excess of activevinyl groups were blocked by 5% mercaptoethanol titrated to pH 9.5 bysodium hydroxide. The gel was washed thoroughly with 1 M sodiumchloride. The binding capacity for wheat germ agglutinin was larger than10 mg lectin per ml gel.

Example 8

Purification of Wheat Germ Agglutinin from a Crude Extract

200 ml of low density conglomerated agarose particles derivatized withdivinyl sulfone and N-acetylglucosamine as described in Example 7 wereused for purification of wheat germ agglutinin from a crude extract. Thebinding of the lectin was performed as an ordinary batch procedurefollowed by collection of the conglomerate particles, washing andelution in a specially developed collection vessel 70 shown in FIG. 7.

"Extraction"

A crude extract of wheat germ was prepared as follows: 1 kg wheat germwas suspended in 20 liters 0.05 M hydrochloric acid at 4° C. Thesuspension was stirred for 4 hours followed by separation of extractfrom germs by crude filtration through a 400 micrometer nylonfilter. Thecrude extract (approx. 15 liters) was adjusted to pH 5.0 with 1 M sodiumhydroxide.

"Adsorption and collection of affinity matrix"

The low density conglomerate agarose N-acetylglucosamine particles weremixed with the crude extract and incubated with stirring for 2 hours.Following binding of the lectin to the conglomerate particles theextract (containing the particles) was pumped through the inlet valve 71of the collection vessel 70. The low density conglomerate particlesraised continuously to the top 75 of the vessel as they entered througha pipe 72 and were then trapped in the vessel (the valve 73 beingclosed), while the extract left the vessel through the outlet valve 74.

"Washing"

After collection of the conglomerate particles in the top section 75 ofthe vessel the vessel and the particles were washed by pumping 0.5 Msodium chloride through the now opened valve 73 in the top and outthrough the outlet valve 74 in the bottom of the vessel (the valve 71being closed). Washing was performed with 5 liters of 0.5 M sodiumchloride.

"Elution"

Elution of the bound wheat germ agglutinin from the conglomerateparticles (still being in the top of the vessel) was performed bypumping ice cold 0.05 M hydrochloric acid into the vessel through valve74 (arrow in parenthesis) in the bottom, up through the bed ofconglomerate particles and out through valve 73 in the top of the vessel75 (the valve 71 being closed and the particles being retained withinthe vessel by the coarse filter 76). The eluted lectin was collected ina total volume of 500 ml 0.05 M hydrochloric acid which was neutralizedby the addition of 1 M dipotassium phosphate. The yield of purifiedwheat germ agglutinin was 360 mg.

Purification of wheat germ agglutinin by traditional packed bedchromatography would require extensive filtration and/or centrifugationas a pretreatment to avoid clogging of the column.

Example 9

Waste Water Treatment Using Immobilized Horse-Radish Peroxidase

Floating immobilized horse-radish peroxidase particles prepared asdescribed in Example 1(e) were then used in a fluid bed for treatment ofindustrial waste water containing a range of phenolic amines andchlorophenols. The untreated waste water was added hydrogenperoxide to aconcentration of 10 mM, pH was adjusted to 5.5 and the waste water wasthen pumped through a down flow fluid bed column containing the floatingimmobilized peroxidase particles stirred proximal to the inlet as shownin FIG. 4. The bed of conglomerate peroxidase particles were dividedinto a mixed zone in the upper part of the fluid bed (upper 7 cm) and azone with particles in a stationary fluidized state (lower 20 cm) bystirring with a velocity of 50 rpm and adjusting the linear flow rate ofthe waste water. The enzymatic oxidation and polymerization of thephenolic compounds caused heavy precipitation in the effluent and aftersedimentation of the precipitate, the content of phenolics in the wastewater had decreased from about 100 ppm to about 10 ppm.

The heavy precipitation of polymerized phenolic compounds would havemade this procedure impossible to perform due to clogging in atraditional packed bed column.

Further more, the use of a stirred fluid bed compared to a non-stirredfluid bed clearly showed less formation of channels through the bed andgave a more complete reaction.

Example 10

Purification of Wastewater from the Fish Industry Using an Ion-ExchangeConglomerate

Untreated wastewater from a fish fillet factory, containing fishproteins, lipids, mucins and other organic compounds was filtered toremove insoluble matter and adjusted to pH 4.5 with hydrochloric acid.

In order to remove the organic contaminants, a 50 liter sample of thewastewater was incubated with 2.5 liters of an acrylic acid copolymericion-exchange conglomerate prepared according to Example 5(d) for 2hours. The incubation was performed as a batch procedure and theconglomerate ion-exchanger was separated from the treated wastewater bypumping it through a collecting vessel as described in Example 8 (FIG.7).

A significant decrease in the content of organic matter after thetreatment was evident from measurements of BOD. BOD values lower than175 mg/l were obtained.

Further more, the ionexchange conglomerate became colored during thetreatment from a white colour to a red-brownish colour.

Example 11

(a) High Density Acrylic Acid Copolymer Solid Class Sphere ConglomeratedParticles

To 300 ml destined water was added 40 ml acrylic acid, 28 g acrylamide,12 g N,N'-methylenbisacrylamide, 5 ml vinyltriethoxysilane and 245 gsolid glass spheres (0.075-0.15 mm, Fryma, Switzerland). The suspensionwas stirred for one hour and then adjusted to pH 7 with cold 27.4%sodium hydroxide. 1 g ammoniumpersulfate and 1 mlN,N,N',N'-tetramethylethylendiamine was added as polymerizationcatalysts and the stirring was continued until a polymer block wasformed. The polymer block was subsequently disintegrated in a blenderfollowed by repeated sedimentation to remove fines. This procedure gaveabout 800 ml conglomerated particles with a density of 1.3 g/ml. In abatch protein binding assay (50 mM Tris/HCl pH 9.0) 1 g of wet butdrained conglomerated particles was able to bind 61% of 190 mg offeredlysozyme from hens egg white.

Selected particles of these conglomerated particles having only onebasic particle are shown in FIG. 1C.

(b) High Density Gelatin Solid Glass Sphere Conglomerated Particles

Four samples of 100 ml 5% gelatin in 0.15 M sodium chloride (35° C.)were added solid glass spheres (0.075-0.15 mm, Fryma, Switzerland) witha density of 2.5 g/ml in increasing amounts:

A: 10 g

B: 50 g

C: 100 g

D: 200 g

After adjustment of pH to 5.5 all samples were added 2.0 ml glutaricdialdehyde (25% solution, Cat. No.: 820603, Merck) under thoroughstirring. After 24 hours of incubation at room temperature thepolymerized matrices were disintegrated in a blender. The resultingparticles were separated from fines by sedimentation. The particles werethen collected on a glass filter and drained for excess water by vacuumsuction on the glass filter. The wet but drained particles were thenweighed and the particle volume determined by adding a known amount ofliquid followed by determination of the total volume. The followingparticle densities were obtained:

    ______________________________________    Measured Density                   Calculated Density    ______________________________________    A:        1.1 g/ml 1.06 g/ml    B:        1.3 g/ml 1.25 g/ml    C:        1.5 g/ml 1.43 g/ml    D:        1.7 g/ml 1.67 g/ml    ______________________________________

We claim:
 1. In a fluidized bed chromatographic process for purificationand binding of molecules in a liquid by binding said molecules to anactive substance covalently bound to chromatographic adsorbent particlesin which process a fluidized bed of said adsorbent particles is formedand said liquid is passed through the fluidized bed, the improvementwherein the chromatographic adsorbent particles comprise a porouscomposite material having pores allowing access to the interior of thecomposite material by said molecules, wherein the porous compositematerial consists of a conglomerate consisting of:(i) at least twodensity controlling particles selected from the group consisting of (a)hollow low density particles which are impermeable to the liquid andhave a density providing floatation of the adsorbent particles in saidliquid, (b) high density particles having a density providingsedimentation of the adsorbent particles in said liquid, and (c) amixture of said low and high density particles; (ii) a matrix formed byconsolidating at least one conglomerating agent selected from the groupconsisting of natural and synthetic organic monomers and polymers; and(iii) an active substance for binding molecules in said liquid;saiddensity controlling particles being dispersed in said matrix, and saidactive substance being covalently bound to said matrix; wherein; saidadsorbent particles have a relative density with respect to said liquidwhich is less than 0.95 or greater than 1.1 and a particle size withinthe range of 50-750 μm, and each of the relative density and particlesize range of said adsorbent particles is selected to provide desiredfloatation/sedimentation properties of said adsorbent particles in theliquid in said fluidized bed process with substantially no turbulence inthe fluidized bed.
 2. The process according to claim 1, wherein thechromatographic process is a procedure selected from the groupconsisting of:ion-exchange chromatography, biospecific affinitychromatography, and group specific affinity chromatography.
 3. Theprocess according to claim 1 or 2, wherein the fluid bed reactor is aliquid down-flow fluid bed reactor comprising a vertical reactor vesselwith an inlet, an outlet, a fluid bed of said chromatographic adsorbentparticles, and agitation means; said agitation means being located nearor in the fluid bed proximal to the liquid inlet.
 4. The processaccording to claim 3, wherein;a) the chromatographic adsorbent particlesand liquid proximal to the liquid inlet are agitated to divide the fluidbed into;i) a turbulent zone having vigorously moving particles, and ii)a non-turbulent zone;said non-turbulent zone adjoining said turbulentzone; and b) the extent of said turbulent zone is determined by a degreeof agitation selected within a range of from;i) a degree of agitationproviding turbulence only in the uppermost part of the fluid bed, to ii)a degree of agitation providing turbulence of the particles throughoutthe fluid bed.
 5. The process according to claim 1, wherein said highdensity particles are impermeable to the liquid.
 6. The processaccording to claim 1, wherein the low density particles are hollow. 7.The process according to claim 1, wherein the high density particles aresolid.
 8. The process according to claim 1, wherein the densitycontrolling particles constitute from 1 to 95%, by volume, of theconglomerate.
 9. The process according to claim 1, wherein the densitycontrolling particles are made of a material selected from the groupconsisting of natural and synthetic organic polymers, and inorganicsubstances and compounds.
 10. The process according to claim 9, whereinthe density controlling particles are made of a synthetic organicpolymer selected from the group consisting of:phenol-formaldehyderesins; ABS resins; polyamides, polyesters, polyethers, polymeric vinylcompounds, polyalkenes, and substituted derivatives thereof; andcopolymers of two or more of said polymers, and substituted derivativesof such copolymers.
 11. The process according to claim 9, wherein thedensity controlling particles are made of one or more inorganicsubstances selected from the group consisting of:anhydrous forms ofsilicon dioxide; metal silicates; metal phosphates; metal oxides; metalsulfides; crystalline forms of carbon; and amorphous forms of carbon.12. The process according to claim 1, wherein said density controllingparticles are made of amorphous silica, quartz, or glass.
 13. Theprocess according to claim 1, wherein the low density particles consistof unicellar glass microspheres.
 14. The process according to claim 1,wherein the high density particles consist of glass microspheres. 15.The process according to claim 1, wherein the at least oneconglomerating agent is made of natural or synthetic organic monomersand polymers selected from the group consisting of:a) natural andsynthetic polysaccharides selected from the group consisting of agar,alginate, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth,karaya gum, locust bean gum, xanthan gum, agaroses, celluloses, pectins,mucins, dextrans, starches, heparins, chitosans, hydroxy starches,hydroxypropyl starches, carboxymethyl starches, hydroxyethyl celluloses,hydroxypropyl celluloses, and carboxymethyl celluloses; b) syntheticorganic polymers and monomers resulting in polymers selected from thegroup consisting of acrylic polymers, polyamides, polyamides,polyesters, polyethers, polymeric vinyl compounds, polyalkenes, andsubstituted derivatives thereof, as well as copolymers comprising morethan one such organic polymer functionality, and substituted derivativesthereof; and c) mixtures of the above.
 16. The process according toclaim 1, wherein the conglomerating agent is agarose.
 17. The processaccording to claim 1, wherein the active substance comprises a materialor mixtures of materials selected from the group consisting of:organicand inorganic compounds or ions.
 18. The process according to claim 1,wherein the active substance comprises a member selected from the groupconsisting of:ligands, charged species for ion exchange chromatography,proteins, dyes, enzyme inhibitors, biotin for purification of avidin andother biotin binding proteins, carbohydrates for purification of lectinsor glycosidases, protein A, chelates, iminodiacetic acid, amino acids,arginine, lysine, and histidine, sulfated polymers, heparins,benzhydroxamic acid, hydrocarbon groups divinyl sulfone activatedsubstances coupled with mercaptoethanol, 4-hydroxypyridine,3-hydroxy-pyridine, or 2-hydroxy-pyridine; natural and syntheticpolynucleotides and nucleic acids; carbohydrate based polymers selectedfrom the group consisting, agar, alginate, carrageenan, guar gum, gumarabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthangum, agaroses, celluloses, pectins, mucins, dextrans, starches andheparins; amino acid based polymers selected from the group consistingof gelatins, albumins, hemoglobulins, immunoglobulins including poly-and mono clonal antibodies, antigenes, protein G, lectins, glycoproteinssuch as ovomucoids, biotin binding proteins, avidin and streptavidin,enzymes, proteases, and protease inhibitors; and mixtures of these. 19.The process according to claim 1, wherein the active substance iscovalently bound to the adsorbent particles by means of activation orderivatization agents effective for activating or derivatizing theconglomerating agent, or the conglomerate particles.
 20. The processaccording to claim 1, wherein the activation or derivatization agentsare selected from a group consisting of:cyanogen bromide, divinylsulfone, epichlorohydrine, bisepoxyranes, dibromopropanol, glutaricdialdehyde, carbodiimides, anhydrides, hydrazines, periodates,benzoquinones, triazines, tosylates, tresylates, and diazoninum ions.21. The process according to claim 1, wherein the relative density isfrom 1.1 to
 5. 22. The process according to claim 1, wherein therelative density is from 0.2 to 0.95.
 23. The process according to claim1, wherein the particle size is within the range of 50-500 μm.
 24. Theprocess according to claim 1, useful for purification and bindingproteins and other high molecular weight substances, wherein theparticle size is within the range of 100-500 μm.
 25. The processaccording to claim 1, wherein the fluid bed reactor is a liquid up-flowfluid bed reactor comprising a vertical reactor vessel with an inlet, anoutlet, a fluid bed of said chromatographic adsorbent particles, andagitation means; said agitation means being located near or in the fluidbed proximal to the liquid inlet.
 26. The process according to claim 25,wherein;a) the chromatographic adsorbent particles and liquid proximalto the liquid inlet are agitated to divide the fluid bed into:i) aturbulent zone having vigorously moving particles, and ii) anon-turbulent zone;said non-turbulent zone adjoining said turbulentzone; and b) the extent of said turbulent zone is determined by a degreeof agitation selected within a range from:i) a degree of agitationproviding turbulence only in the lower-most part of the fluid bed, toii) a degree of agitation providing turbulence of the particlesthroughout the fluid bed.
 27. The process according to claim 1 whereinthe density controlling particles constitute 5-30% by volume of theconglomerate.